Disruption of the pro-oncogenic c-RAF–PDE8A complex represents a differentiated approach to treating KRAS–c-RAF dependent PDAC

Pancreatic ductal adenocarcinoma (PDAC) is considered the third leading cause of cancer mortality in the western world, offering advanced stage patients with few viable treatment options. Consequently, there remains an urgent unmet need to develop novel therapeutic strategies that can effectively inhibit pro-oncogenic molecular targets underpinning PDACs pathogenesis and progression. One such target is c-RAF, a downstream effector of RAS that is considered essential for the oncogenic growth and survival of mutant RAS-driven cancers (including KRASMT PDAC). Herein, we demonstrate how a novel cell-penetrating peptide disruptor (DRx-170) of the c-RAF–PDE8A protein–protein interaction (PPI) represents a differentiated approach to exploiting the c-RAF–cAMP/PKA signaling axes and treating KRAS–c-RAF dependent PDAC. Through disrupting the c-RAF–PDE8A protein complex, DRx-170 promotes the inactivation of c-RAF through an allosteric mechanism, dependent upon inactivating PKA phosphorylation. DRx-170 inhibits cell proliferation, adhesion and migration of a KRASMT PDAC cell line (PANC1), independent of ERK1/2 activity. Moreover, combining DRx-170 with afatinib significantly enhances PANC1 growth inhibition in both 2D and 3D cellular models. DRx-170 sensitivity appears to correlate with c-RAF dependency. This proof-of-concept study supports the development of DRx-170 as a novel and differentiated strategy for targeting c-RAF activity in KRAS–c-RAF dependent PDAC.

Due to the disordered nature of RAF kinase termini, purification and generation of full-length RAF proteins are highly challenging.binds, with graphical representation of assay to right of graph.(A)(ii) Representative human c-RAF peptide array (20mers) highlighting primary and secondary bind regions and (A)(iii) proposed PDE8A1 primary binding region superimposed onto a 3D c-RAF kinase domain structure (PDB: 3OMV 28 ), located at a site within c-RAF's C-Lobe and removed from the active ATP binding site and dimerisation interface.(B)(i) Representative coomassie and immunoblot of GST and catalytically 'active' c-RAF kinase domain (KD: S306-F648, Y340D/Y341D)-GST proteins.(B)(ii) DRx-170F disruptor peptide (blue, N = 5), but not DRx-150F negative control peptide (grey, N = 4), directly binds c-RAF(KD)-GST protein (MEAN ± SEM, Kd = 1.66 ± 0.15 µM).KD kinase domain.

Afatinib enhances DRx-170 potency in a 2D and 3D human KRAS MT PDAC model
Utilising an existing pancreatic cancer clinical pathology data set, derived from The Human Protein Atlas and The Cancer Genome Atlas (Supplementary Data Set 1), we sought to determine how pancreatic cancer patient survival correlated with EGFR-RAS-RAF mRNA expression.Notably, high EGFR, ERBB2, ERBB3, KRAS, NRAS and c-RAF (but not PDE8A, HRAS, A-RAF or B-RAF) mRNA expression correlated unfavourably with patient survival.Of the limited 176 patients in this data set, 78 (44.3%) expressed high c-RAF mRNA levels.Of these 78 patients, all (100%) expressed high KRAS and/or NRAS mRNA levels.Moreover, 92.3% (72/78) of these patients also expressed high EGFR and/or ERBB2 and/or ERBB3 mRNA levels.Therefore, high c-RAF mRNA levels strongly correlate with KRAS/NRAS and EGFR/ERBB2/ERBB3 mRNA expression.These findings appear consistent with pre-clinical studies supporting a potentially synergistic approach to treating KRAS MT pancreatic adenocarcinomas, through dual inhibition of c-RAF and EGFR 16 .Consequently, we evaluated the combined efficacy of DRx-170 with a 2nd generation (irreversible) EGFR/ERBB-family inhibitor, afatinib (Fig. 4).
Noticeable differences in DRx-170 potency were observed in 2D vs. 3D PANC1 cellular models.Consequently, we sought to determine if this was attributed to DRx-170's duration of activity and/or stability (Fig. S4).Firstly, DRx-170 activity was assessed in the context of low (2%) and high (10%) FBS vs. 2D PANC1 cells (Fig. S4A).No significant difference in PANC1 growth inhibition was observed (Fig. S4A).However, DRx-170's ability to suppress the relative rate of PANC1 growth (i.e., % slope of nCI curve) significantly reduced after initial 24 h treatment period; with PANC1 growth accelerating ~ 24 h post-treatment (Fig. S4A).In addition to relative serum stability, DRx-170 appeared stable in both (in vitro) rat plasma (T ½ > 180 min) and rat liver microsomes (estimated Clint < 13.2 µL/min/mg protein) (Fig. S4B).Moreover, DRx-170 activity was highly comparable regardless of whether it was synthesised as a TFA, chloride or acetate counterion (Fig. S4C).These findings demonstrate that, in these in vitro contexts, DRx-170 stability (and therefore activity) persists for up to 24 h.Whether increased treatment frequency (e.g., every day; q.d.) would enhance 3D PANC1 spheroid growth inhibition was not assessed in this study.

DRx-170 attenuates PDAC cell adhesion and migration
RTCA xCELLigence technology measures a combination of cell proliferation, adhesion, and cell size.To further investigate the observable differences in 2D vs. 3D potency (Fig. 4.), we assessed DRx-170's influence on PANC1 cell morphology and adhesion (Fig. 5).Firstly, through measuring individual cell area, DRx-170 [1 µM] was shown to have no effect on PANC1 cell size (Fig. 5A).PANC1 adherence was then measured using the RTCA xCELLigence platform.PANC1 cells adhere rapidly (approximately 4-6 h 40 ) and their population doubling time is slow (approximately 52 h, ATCC: CRL-1469).Thus, initial increases in cell index (measured via RTCA  www.nature.com/scientificreports/xCELLigence platform) are largely indicative of cellular adherence, not proliferation.Resultingly, relative PANC1 adherence was assessed over an 8 h period following simultaneous cell seeding and treatment (Fig. 5B).DRx-170 induced potent inhibition of relative PANC1 adhesion (Fig. 5B).DRx-150 did not negatively impact adhesion (Fig. 5B).In addition to the already observed anti-proliferative actions of DRx-170 (Fig. 4), these results demonstrate DRx-170's ability to also inhibit PANC1 cell adhesion.Therefore, differences in DRx-170 activity in 2D vs. 3D PANC1 cell models is likely attributed to its additional anti-adhesive activities, and not a change in cell size.
Out with this study, PDE8A and c-RAF have been shown to play an important role in regulating cell adhesion and migration 39,[41][42][43][44] .Resultingly, we investigated DRx-170's ability to influence PANC1 cell migration utilising an in vitro wound healing assay (Fig. 5C).DRx-170, but not DRx-150, significantly suppressed PANC1 cell migration over a 24 h treatment period (Fig. 5C).These findings demonstrate how DRx-170 mediated disruption of the c-RAF-PDE8A PPI not only attenuates PANC1 cell proliferation, but also PANC1 cell adhesion and migration.DRx-170's anti-proliferative activity does not extend to non-cancerous HEK293 (epithelial) or IMR-90 (fibroblast) human cell lines, as seen by no significant change in relative cell viability (Fig. 5D).

DRx-170 activity appears to correlate with KRAS-c-RAF dependency
Harnessing publicly available DepMap portal data sets (https:// depmap.org/ portal/), we interrogated the correlation between RAS-RAF-PDE8A mutational status and c-RAF genetic dependency; determined through genome-scale CRISPR-Cas9 gene essentiality screening (Fig S5, Supplementary Data Set 2).Combined analysis of n = 1079 human cancer cell lines (> 25 lineages) showed that activating mutations in KRAS, NRAS and HRAS correlate significantly with increased c-RAF gene dependency vs. wild-type RAS (Fig. S5A, C-E).No significant difference was observed with PDE8A gene dependency (Fig. S5B).Cancer cell lines harbouring a c-RAF mutation or a non-V600 B-RAF mutation (not an A-RAF mutation), were significantly more dependent upon c-RAF than those that harbour a BRAF V600 mutation (Fig. S5F).Furthermore, PDE8A mutational status does not correlate with c-RAF gene dependency (Fig. S5G).Thus, RAS, c-RAF, and non-V600 B-RAF mutations correlate with increased c-RAF genetic dependency in this data set.

Discussion
Remodelling of the PPI oncoproteome with highly selective peptide-based disruptor therapeutics represents a rapidly emerging strategy to fine-tuning the disease microenvironment in cancer, significantly de-risking the potential for off-target toxicity [45][46][47][48][49] .This next-generation of targeted therapeutics is making significant progress within the clinical setting (e.g., C/EBPβ dimerisation inhibitor-ST101: 49 ) and adds to the growing arsenal of precision medicine tools in oncology research.Given the abysmal survival rate of PDAC, coupled with the severe lack of viable treatment options, novel therapeutic strategies capable of effectively treating this lethal malignancy remain a clear and urgent unmet need.Selectively inhibiting c-RAF's activity (kinase dependent and independent) represents a persistently attractive therapeutic approach in oncology, offering a high efficacy-low toxicity strategy to treating PDAC and other related RAS MT -c-RAF driven malignancies (e.g., lung, colorectal, ovarian, urothelial, and skin cancer).Utilising our first in class cell-penetrating disruptor peptide therapy (DRx-170), we present proof-of-principle data which demonstrates that selective disruption of the pro-oncogenic c-RAF-PDE8A PPI is an encouraging and differentiated approach to inhibiting human KRAS-c-RAF dependent PDAC cell proliferation, adhesion, and migration (Fig. 7).
Through regulating the cAMP microdomain surrounding c-RAF, PDE8A plays a central role in managing the crosstalk between the c-RAF and cAMP-PKA signaling axes [24][25][26] .Given that c-RAF binds PDE8A at a conserved region upstream of its catalytic domain 26 , it could be suggested that c-RAF can bind all PDE8A1-5 isoforms.Although the functional relevance of this is yet to be discovered, it is highly likely that PDE-specific compartmentalisation plays a significant role in dictating which PDE8A isoforms bind c-RAF, and at which location(s) within the cell this occurs 21,22 .Conversely, protein sequence identity analysis of PDE8A1 binding sites on c-RAF (Fig. 1, Fig. S2) highlight a high degree of sequence conservation within the kinase domain and C-terminus of A-RAF and B-RAF.Though no findings to date have identified PDE8A as a binding partner of A-RAF or B-RAF, our data suggests there is potential for PDE8A to bind all RAF isoforms.Again however, Vol:.( 1234567890 PDE8A isoform compartmentalisation, along with c-RAF-PDE8A localisation and the relative prevalence of RAF homo/heterodimers will presumably direct this.In the context that PDE8A exclusively binds c-RAF, it could be assumed that disrupting the c-RAF-PDE8A PPI with DRx-170 has the potential to regulate all RAF isoforms present within a given RAS-RAF signalosome.Thus, characterising isoform specificity will be a central theme of future research focused on elucidating DRx-170's mechanism. As previously documented, disrupting the c-RAF-PDE8A PPI upregulates inhibitory PKA-mediated phosphorylation of c-RAF at serine 259 (S259) 26,39 , a direct indication that c-RAF is in a non-active closed conformation via a 14-3-3 dependent mechanism [33][34][35][36][37] .Following an increase in S259 phosphorylation, upregulation of serine 43 (S43) phosphorylation was observed (Fig. 3).S43 represents another well characterised inhibitory PKA phospho-site on c-RAF, that when phosphorylated stoichiometrically hinders RAS from binding c-RAF's RBD (Ras Binding Domain) [32][33][34][35][36][37] .These data suggest (for the first time) that targeted c-RAF-PDE8A disruption promotes RAS-RAF dissociation.Given that RAS-RAF complex formation significantly enhances RAF dimer formation, future studies will look to determine how DRx-170 influences RAF homo/hetero-dimerisation.Moreover, broader profiling of c-RAF's inhibitory PKA-phospho sites (including S233 and S621) will need carried out to fully characterise their utility as a c-RAF specific biomarker panel consistent with DRx-170 activity.Though it remains a poorly defined area of research, characterisation of corresponding A-RAF (S214, S582) and/or B-RAF (S365, S729) phospho-sites may prove useful in said biomarker panel, and thus remains an area of interest [50][51][52] .
As anticipated, no change in ERK1/2 activity was observed following c-RAF-PDE8A disruption (Fig. 3).This is in line with pre-clinical studies highlighting a non-essential role for c-RAF kinase-dependent signaling in promoting KRAS MT PDAC [13][14][15][16][17] .Thus, data suggests DRx-170 inhibits KRAS MT PANC1 cell growth, adhesion, and migration independent of c-RAF's catalytic activity.Research associated with c-RAF's kinase-independent mechanisms remain within in its infancy.Existing studies have highlighted several c-RAF PPIs that promote cancer cell survival through suppression of pro-apoptotic signaling (e.g., ASK1, MST2, Bcl-2) and regulation of cell motility/de-differentiation (e.g., ROKα) [10][11][12] .c-RAF's role in promoting STAT3 activation has also been associated in PDAC and colorectal cancer as being pro-oncogenic, irrespective of c-RAF kinase activity 16,53 .Furthermore, disruption of the recently characterised HSP90-CDC37-c-RAF complex highlights yet another approach to de-stabilising c-RAF 54 .Although these discoveries provide rational direction for future mechanism/biomarker stratification research in RAS MT cancer, it is important to acknowledge both the significant size of the RAF PPI interactome [55][56][57] , and the fact that c-RAF can promote cancer cell proliferation independent of RAS 53 .Thus, careful elucidation of mechanism(s) is crucial to further differentiating DRx-170 from existing RAF inhibitors.
Precision medicine remains at the forefront of experimental and clinical oncology, supporting the development of targeted therapeutics through accurately characterising each patient's cancer into specific actionable sub-types.This is also true for PDAC, where the median overall survival of PDAC patients who received genomically matched treatment regime(s) was significantly longer than those who did not (~ 1.71 to 1.96-fold longer) 5,58 .Of relevance, PDAC biomarker signature studies have robustly demonstrated a correlation with DNA-damage response, replication stress, and receptor tyrosine kinase enrichment with platinum-based/PARP inhibitors, cellcycle inhibitors, and EGFR/ERBB-family inhibitors respectively 59,60 .Thus, to foster the successful pre-clinical and clinical development of our c-RAF-PDE8A disruptor therapeutic, it is critical that we clearly categorise biomarkers that: (i) elucidate the mechanism(s) associated with PPI disruption, (ii) allow for prediction of treatment sensitive vs. resistant cancer models, and (iii) highlight rational combination therapy strategies that can overcome potential acquired resistance.Our preliminary findings suggest that future investigations should www.nature.com/scientificreports/be inclusive of RAS-RAF mutational status (excluding B-RAF V600 mutations) and associated dependencies in the EGFR/ERBB-RAS-RAF signaling pathway.

Peptide array epitope mapping
Peptide array experiments were performed by automatic SPOT synthesis as described 26,27 .Human c-RAF peptides were synthesised onto PEG-derivatized continuous cellulose membrane supports via 9-fluorenylmethyloxycarbonyl chemistry (Fmoc) using the MultiPep 2 Robot (CEM).A far western blot approach was utilised to detect PDE8A1-MBP binding, where-by c-RAF arrays (consisting of 20mer peptide fragments overlapping by five amino acids) were (i) blocked for 1 h at room temperature in 2.5% milk in 1 × TBS, (ii) incubated overnight at 4 °C in [0.1 μM] PDE8A1-MBP (diluted in 1 × TBS, 5% glycerol, pH 7.4), (iii) incubated for 1 h at room temperature in αMBP-HRP primary antibody (1:1000, Abcam: ab49923) and (iv) visualised via ECL detection utilising the C-Digit Blot Scanner (LI-COR).Arrays were washed three times in 1× TBS-T following primary and secondary antibody incubation steps.MBP (i.e., myosin binding protein) alone was used as a negative protein control.PDE8A1-MBP and MBP proteins are described previously 26 .

Fluorescent ligand-based binding assay
Glutathione coated wells of a pre-blocked, black, clear bottom, 96-well plate (#15340, Thermo) were incubated with 50 ng of c-RAF kinase domain protein (#14-352, Merck) or GST protein (gifted by Prof. George Baillie) and incubated overnight at 4 °C.Wells were then incubated with increasing concentrations of FITC-labelled peptide [0.6-10 μM] for 2 h at room temperature.Excess protein/peptide was removed following each incubation step by washing three times in 1× TBS-T.Protein and peptides were diluted in binding buffer (200 mM NaCl, 50 mM Tris, 5 mM DTT, 5% Glycerol, protease cocktail inhibitor tablet (Roche), pH 7.5).FITC-peptide binding to c-RAF protein was measured using a Tristar 5 multimode microplate reader (Berthold Technologies).Binding affinities were measured via non-linear regression analysis (GraphPad Prism 8.0).

Immunocytochemistry (ICC)
PANC1 cells were seeded at 0.5 × 10 5 cells per well of a 12-well plate containing a sterilised 0.13-0.17mm glass coverslip in complete DMEM and incubated overnight.Cells were fixed in 4% paraformaldehyde (Sigma) for 15 min at room temperature.Cell membranes were permeabilised with 0.1% Triton × 100 (sigma) for 4 min at room temperature.Cells were then blocked for 1 h at room temperature with 10% donkey serum, 1% BSA in PBS.Both PDE8A (rabbit) and c-RAF (mouse) primary antibodies were then diluted 1:100 in 5% donkey serum, 1% BSA in PBS and cells incubated overnight at 4 °C.Secondary Alexa Fluor antibodies were then simultaneously incubated for 1 h at room temperature.Cells were washed three times in PBS between each of the above steps.Finally, coverslips were then mounted onto glass slides with Prolong Gold Antifade Mountant with DAPI (Thermo, P36941).In experiments where PDE8A protein expression was assessed following treatment, PANC1 cells were treated appropriately prior to fixation.PANC1 cells were imaged using a Zeiss (LSM880) confocal microscope.

Western immunoblotting
Protein lysates were harvested using lysis buffer (25 mM Tris, 150 mM NaCl, 0.1 mM EDTA, 1% NP-40, 5% glycerol, pH 7.4) supplemented with protease and phosphatase inhibitors (Roche).Protein samples were diluted in SDS sample buffer (10% SDS, 300 mM Tris-HCl, 0.05% bromothymol blue, 10% β-mercaptoethanol) and boiled for 10 min at 70 °C.Proteins were resolved via SDS-PAGE using 4-12% Bis-Tris gels (NuPAGE), transferred to nitrocellulose membranes (GE Healthcare).To allow for simultaneously incubation of more than one antibody with a single membrane, full membranes were cut at appropriate molecular weight markers (see Supplementary ' Appendix 1.1-All Immunoblots' for full immunoblots and replicates).Membranes were blocked in Intercept TBS blocking buffer (LI-COR) and incubated overnight in primary antibody at 4 °C.IRDye secondary antibody (1:10,000, LI-COR) was then incubated for 1 h at room temperature and immunoreactive bands visualised using the Odyssey CLx imaging system (LI-COR).Densitometry of immunoreactive bands was carried out using Image J software.All proteins were normalised to their respective housekeeper protein (HSP90) or Revert ™ 700 Total protein stain (LI-COR, 926-11011).Phosphorylated proteins were normalised to their respective total.

Real-time cellular analysis xCELLigence assay
Label-free cellular growth of human cancer cell lines were measured using the xCELLigence real-time cellular analysis platform (RTCA, Roche Applied Science) as per manufacturer's instructions.96-well E-plates were utilised to measure cellular impedance within each well, providing quantitative measurements associated with cell proliferation, adherence, and morphology (represented as cell index (CI)).To assess the influence of c-RAF-PDE8A disruption on cancer cell growth, cells were seeded at 1 × 10 4 cells per well.Following complete PANC1 cell adherence (approximately 4-6 h 40 ), cells were treated with the appropriate concentration of drug(s) for 24-60 h and CI monitored every 15 min.To assess PANC1 cell adherence, cells were seeded at 2 × 10 4 cells per well and immediately treated with appropriate concentration of drug for 8 h (CI measured every 5 min).Unless otherwise stated, all treatments were carried out in respective media containing 2% FBS and to a final DMSO concentration of ≤ 1%.CI was normalised to 1 at treatment timepoint and the rate of growth (i.e., slope of CI curve) analysed via linear-regression analyses (GraphPad Prism 8.0).

3D-spheroid growth assay
PANC1 cells were seeded at 2 × 10 3 in wells of a round bottom Nunclon™ Sphera™ 96 well-plate with ultra-low attachment coating (Thermo; #174925), containing 200 μL DMEM (2% FBS, 2 mM l-glutamine, 100 U/I Pen-Strep).To encourage spheroid formation, PANC1 cells were centrifuged at 250 × g for 10 min at room temperature.PANC1 spheroids were then allowed to grow for 3 days at 37 °C, 5% CO 2 , humidified air.Spheroids were then treated with appropriate concentration of DRx-peptide/afatinib by first removing 100 μL of media from each well (careful not to disturb spheroid), followed by addition of 100 μL drug at 2× concentration.Cells were treated five times over a 10 day period (day 0, 2, 4, 7 and 9).Spheroids were imaged on day 0, 2, 4, 7 and 10 using a Nikon Eclipse TS2 microscope and spheroid area quantified using Image J software.Data represented as a fold-difference of relative day 0 spheroid measurement (i.e., day 0 normalised to 1).

PANC1 cell area
PANC1 cells were seeded at 0.5 × 10 5 cells per well of a 6-well plate in complete DMEM and incubated overnight.PANC1 cells were then treated for 24 h with vehicle (0.5% DMSO), DRx-170 [1 µM] or left untreated.Following treatment, cells were fixed in 4% paraformaldehyde (Sigma) for 15 min at room temperature.Nikon Eclipse TS2 microscope was used to image PANC1 cells and cell area quantified using Image J software.

In vitro scratch-wound healing assay
PANC1 cells were seeded at 3 × 10 5 in wells of a 24 well plate and allowed to grow overnight in DMEM (10% FBS, 2 mM l-glutamine, 100 U/I Pen-Strep).Confluent PANC1 monolayer was then manually 'scratched' using a sterilised p200 pipette tip.Cells were then washed two times with PBS to remove detached cells.Cells were then incubated in DMEM (2% FBS, 2 mM l-glutamine, 100 U/I Pen-Strep) containing appropriate concentration of DRx-peptide or vehicle (1% DMSO) and denuded area was immediately imaged (brightfield) using a Nikon Eclipse TS2 microscope.'Wound' was imaged at 24 h post-treatment.The area of 'wound' was quantified using the 'Wound Healing Size Tool' plugin on Image J software and normalised as % gap closure to respective 0 h measurement 61 .

Figure 7 .
Figure 7. Schematic illustrating how DRx-170 binds c-RAF, displaces PDE8A and exposes c-RAF to surrounding cAMP microenvironment in the context of KRAS MT cancer.De-protection negatively regulates c-RAF activity in a PKA-dependent manner (pS43/pS259 validated, pS233/pS621 untested), promoting c-RAF conformational closure and dissociation from upstream KRAS.This conservative model depicting DRx-170 mechanism of action highlights how DRx-170 attenuates tumourigenesis through facilitating the allosteric inhibition c-RAF.RBD ras binding domain; CRD cysteine rich domain; AC adenylate cyclase; cAMP cyclic adenosine monophosphate; ATP adenosine triphosphate; AMP adenosine monophosphate; PKA protein kinase A.